Apparatus for red blood cell separation

ABSTRACT

Red blood cells are removed from whole blood or a fraction thereof by contacting whole blood with a combination of an agglutinating agent and nucleating particles to form clusters of red blood cells. High molecular weight polyethylene glycol may be added further to enhance agglutination. The clusters of red blood cells are much larger than the size of individual red blood cells, so that the clusters can easily be filtered through a porous medium. The plasma which is substantially free of red blood cells is further passed through a filter that optionally contains an additional agglutinating agent. Flow-delay means may be provided to return the fluid sample in contact with a regard for a predetermined time.

This is a division of parent application Ser. No. 08/203,778 filed Mar.1, 1994, now abandoned which is a continuation-in-part of Ser. No.08/049,862, filed on Apr. 20, 1993, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method and apparatus for separatingred blood cells from whole blood.

The present application is related to application Ser. No. 08/049,862,filed Apr. 20, 1993, the entire contents of which are herebyincorporated by reference.

2. Description of the Background Art

The ability to measure a wide variety of physiologically activecompounds, both naturally occurring and synthetic, has become ofincreasing importance, as an adjunct to both diagnosis and therapy.While for the most part such assays have required clinical laboratorydeterminations, there is an increasing awareness of the importance ofbeing able to conduct assay determinations in a physician's office or inthe home. These environments require that the assay have a simpleprotocol and be relatively free of sensitivity to small changes in theconditions under which the assay is carried out. Importantly, inaccuratemeasurements of reagents and sample should, whenever feasible, beavoided. A number of systems have been developed to address the variousproblems associated with analysis outside of the clinical laboratory.

One analyte of importance is cholesterol. There is a clearly establishedrelationship between total blood cholesterol (mainly the LDL fraction)and coronary artery disease (J.A.M.A. 253: 2080-2086, 1985). Newguidelines have been established for adults to identify risk groupsassociated with blood cholesterol levels. Since cholesterol levels canbe controlled by both diet and cholesterol lowering drugs, for thoseindividuals at risk, the ability to monitor one's own cholesterol athome is useful in reducing the potential for heart disease. Themeasurement of other naturally occurring compounds of physiologicalimportance, such as glucose, lipoproteins, etc., as well as syntheticdrugs, is also of great interest, as assays become more sensitive andcontrol can be exercised more diligently by the patient.

In clinical assays, the separation of serum or plasma from whole bloodis extremely important, since it is often difficult to conduct theanalysis of dissolved blood components without interference from the redblood cells. Serum or plasma is conventionally separated fromerythrocytes by centrifuging. Centrifugation, however, causes otherproblems because one must then separate the supernatant from the bloodcake. Moreover, this method is not available for use in home or officediagnostic assays.

Using whole blood with diagnostic devices for use in home or officeassays gives rise to further problems. In these devices, it is customaryto employ reagents which cause a color change if the analyte is present(or, alternatively, if it is absent). Turbid or colored solution, suchas whole blood, may interfere with the readings.

Means for the fractionation of whole blood into blood cell plasmafractions are known in the art.

Vogel et al., in U.S. Pat. No. 4,465,24, disclose a process forseparating plasma or serum from whole blood using a filter made of glassfibers. The glass fibers used have an average diameter of 0.2 to 5microns, and a density of about 0.1 to 0.5 g/cm. Whole blood is placedonto a layer of glass fibers, and plasma is generated by retardation offlow of the cells. Plasma is collected at the other side of the glassfibers.

Another approach to separating red blood cells from whole blood is shownin Hillman et al., U.S. Pat. No. 4,753,776. In this patent, capillaryaction is used to pull whole blood through a glass microfiber filter byretarding the flow of the cells.

Allen et al., in U.S. Pat. No. 4,987,085, disclose a device and methodfor separating plasma from whole blood via a filtering system withdescending pore size to provide for successive removal of red bloodcells without lysis. A combination of glass fiber membranes andcellutosic membranes is used to minimize red blood cell lysis whileremoving red blood cells.

Kondo et al., in U.S. Pat. No. 4,256,693, disclose a multilayeredintegral chemical analysis element for blood comprising a filter layercapable of removing formed components from the blood. The filter layermay be made of at least one component selected from paper, nonwovenfabric, sheet-like filter material composed of powders or fibers such asman-made fibers or glass fibers, and membrane filters having suitablepore sizes. The filter layer separates the formed components of theblood at one time, or successively, such as in the order of leukocytes,erythrocytes, and platelets.

Other filtration systems are described in U.S. Pat. Nos. 3,092,465;3,630,957; 3,663,374; 4,246,693; 4,246,107; and 2,330,410. Some of thesefilters employ membranes with small pores. A disadvantage of thesefilters is that blood can only penetrate through the membrane filtervery slowly and in small amounts, because the membrane is very easilyblocked. This results in a reaction which takes longer than isdesirable.

Unfortunately, blood separation devices using glass fiber filters ormembranes tend to retain significant amounts of serum or plasma, anddisplay a relatively slow speed of separation. This presents a problemwith diagnostic devices which are quantitative, as there must besufficient sample present in the detection area to provide a reliableindication of the quantity of analyte detected. If insufficient sampleflows through the filter, a false low reading will be obtained.Moreover, devices intended for home or office use should be convenientto use and should provide an indication of the analyte within areasonably short period of time. It is thus essential to remove unwantedred blood cells to allow most of the remainder of the blood to passthrough the separation device, and then filter the blood relativelyquickly.

To solve these problems, test papers have been coated withsemi-permeable membranes (U.S. Pat. No. 3,092,465), and swellable filmsinto which only the dissolved components of the blood can penetrate,leaving the erythrocytes (U.S. Pat. No. 3,630,957). These two methodsare only useful for testing low molecular weight components of bloodsuch as glucose or for urea. Higher molecular weight components of theblood such as lipids, or substrates bound to serum protein, such asbilirubin, cannot be determined in this way because they are not able topenetrate into the film or to pass through the semipermeable membrane.

Alternative solutions include covering diagnostic agents with membranefilters for separating the blood cells, as disclosed in U.S. Pat. Nos.3,663,374 and 4,246,693. A disadvantage with these diagnostic agents isthat the blood can only penetrate through the membrane filter veryslowly and in small amounts, because the membrane is very easilyblocked. This results in a reaction which takes longer than isdesirable.

U.S. Pat. Nos. 4,246,107 and 2,330,410, teach that lymphocytes andleukocytes can be separated from blood when the blood is filteredthrough a layer of synthetic resin fibers with an average fiber diameterof from 5 to 20 microns for lymphocytes, and from 3 to 10 microns forleukocytes. However, since the erythrocytes preponderantly pass throughthe filter with the plasma, these filters are not suitable for obtainingplasma.

Red blood cells can also be removed from whole blood samples bycontacting a whole blood sample with an agglutinating agent. One type ofred blood cell agglutinating agent is a lectin, a family of sugarbinding protein first identified in plants. Red blood cells can beremoved from whole blood by contacting the whole blood with a lectin,which attaches itself to specific glycoproteins on the red blood cellmembrane and forms large masses by agglutination of the cells. However,when lectins contact red blood cells, cross-linking occurs to form agel. The presence of this gel in a filter greatly reduces the flow ofblood through a filter, and therefore limits the amount of plasmarecovered.

Sand et al., in U.S. Pat. No. 5,118,428, disclose using an acid, such ascitric, acetic or ascorbic acid, to agglutinate the red blood cells forseparation from whole blood. However, use of an acid lowers the pH ofthe plasma, which may interfere with subsequent analyses.

Trasch et al., in U.S. Pat. No. 5,055,195, disclose the use oferythrocyte-retention substrates which contain two strongly polar groupswhich are connected by a non-polar bridge which serves as a spacer.These substrates change the polarity of the surface of the erythrocytesand cause them to agglutinate. The agglomerates formed in the blood canthen be readily separated by filtration.

Zuk, in U.S. Pat. No. 4,594,327, teaches a method for separating redblood cells from whole blood by combining the whole blood sample with ared-blood cell binding agent. This mixture is then filtered through asolid bibulous agent.

Rapkin et al., U.S. Pat. No. 4,678,757, disclose a method for separatingblood into fluid and cellular fraction for diagnostic tests. The wholeblood is introduced into a carrier containing a layer of carbohydrate,which separates the fluid from the cellular fractions.

Hillman et al., in U.S. Pat. Nos. 4,753,776 and 5,135,719, disclose amethod for separating plasma from red blood cells wherein a low pressurefilter is interposed in a pathway between an inlet port and a reactionarea. Capillary force is the sole driving force for the movement ofplasma from the filter to the reaction area. The filter is made of glassmicrofiber filters which can operate in the presence or absence oragglutins.

A device for separating plasma or serum from whole blood is disclosed inAunte et al., U.S. Pat. No. 4,933,297. This device consists of a matrixof hydrophilic sintered porous material with at least one red blood cellagglutinating agent incorporated therein. An optional filter containingthe same agglutinating agent is added to give a filter combination thatyields plasma which is about 97% free of red blood cells.

Laugharn et al., in U.S. Pat. No. 4,946,603, describe a filter means toretain blood cells which pass through a matrix of hydrophilic sinteredporous material to which at least one red blood cell agglutinating agenthas been applied. The agglutinating agents used include natural andsynthetic water soluble polymers, including hexadimethriine bromide,polylysine, and anti-red blood cell antibodies. The agglutinationprocess is enhanced by incorporating substances such as polyvinylpyrrolidone which functions as a dielectric, allowing charged cells toapproach one another and by crosslinking by antibody and/or otheragglutinins.

Many of the most commonly used assays in disposable assay devicesrequire an incubation step, such as requiring enzymes to act on thesample, such as determinations of cholesterol, glucose, uric acid, andthe like. Additionally, enzymes are often used as labels in immunoassay.In a conventional enzyme immunoassay, an enzyme is covalently conjugatedwith one component of a specifically binding antigen-antibody pair, andthe resulting enzyme conjugate is reacted with a substrate to produce asignal which is detected and measured. The signal generated by theenzyme, in either the conventional or the immunoassay, may be a colorchange, detected with the naked eye or by a spectrophotometrictechnique.

However, despite many attempts by prior workers to provide means toseparate red blood cells from whole blood quickly and efficiently, thereare still many disadvantages to the methods described above.

Ideally, a disposable assay device should include a means to delay theflow of the sample through the device for a predetermined time to permitincubation of the sample with the reagents or indicators present in aparticular region of the device. After the incubation period, which isgenerally on the order of a few minutes or less, the sample then flowsto the next region of the device for further processing. An ideal flowdelay means should work like a valve, with a "closed" and an "open"state. When the state is "closed", the fluid flow should stop, and whenthe state switches to "open", the fluid should flow through theflow-delay valve with little or no restriction, and the flow rate of thefluid through the device should be substantially unchanged.

A wide range of disposable analytical devices have been developed whichinclude means to control flow of fluids therethrough. However, none ofthese previously developed devices has a valve-like means to control theflow of fluids.

Although there has been no previous disclosure of assay devicesincluding a valve-like means, a number of disposable analytical devicesinclude means for delaying flow. Examples of these can be found inDeutsch et al., U.S. Pat. No. 4,522,923; Ebersone, U.S. Pat. No.4,522,786; Jones, U.S. Pat. No. 5,213,965; Vonk, U.S. Pat. No.5,185,127.

Assay devices in which contact between layers is delayed by physicallyseparating the layers until the operator places the layers into contactwith each other are shown in Deneke et al., U.S. Pat. No. 4,876,076; andRamel et al., U.S. Pat. No. 4,959,324.

Other means for controlling flow of samples through assay devicesinclude those of Woodrum, U.S. Pat. No. 4,959,305, reversiblyimmobilized assay reagents; Columbus, U.S. Pat. No. 4,549,952, viscosityincreasing means; Bruce et al., Analytical Chemica Acta 249 (1991),263.269, expansion of compressed foam; Hillman et al., U.S. Pat. No.4,963,498, agglutination of binding pairs; Kurn et al., U.S. Pat. No.5,104,812, interrupting capillary flow.

Physical barriers to interrupt flow are shown in Columbus, U.S. Pat.Nos. 4,310,399 and 4,618,476 and Grenner et al., U.S. Pat. No.5,051,237.

Liquid flow through a filter is controlled by reactions between thesample and a component of the filter, such as in Marchand et al., U.S.Pat. No. 5,127,905 and Tanaka et al., U.S. Pat. No. 4,966,784.

Impregnated layers which are not valve-like in their actions are shownin Liotta, U.S. Pat. No. 3,723,064; Engelmann, U.S. Pat. No. 4,738,823;Nagatomo et al., U.S. Pat. No. 4,587,102; Koyama et al., U.S. Pat. No.4,615,983; Rothe et al., U.S. Pat. No. 4,587,099; Nelson, U.S. Pat. No.4,923,680.

Moreover, none of the above-noted patents provides a reliable means formetering the rate of flow delay through a layer in order to retain asample in contact with a reagent for a predetermined length of time.

No admission is made that any of the patents or other background artcited above constitutes prior art.

SUMMARY OF THE INVENTION

The present invention provides an efficient and rapid means for removingred blood cells from a blood sample. This method can be used inconjunction with an assay device for use in a physician's office or thehome, as the red blood cells are rapidly removed from whole blood whilemost of the serum or plasma passes rapidly through the device forsubsequent analysis.

The device of the present invention provides cluster forming means madeof a porous material such as an absorbent pad which contains thereinboth agglutinating agents and nucleating particles. These agglutinatingagents and nucleating particles cooperate synergistically to rapidlyagglutinate red blood cells, allowing high flow rates through this partof the device while still achieving a substantially complete removal ofred blood cells. The agglutinating agent can be conjugated to thenucleating particles, or can be mixed with the nucleating particles. Redblood cells are agglutinated and trapped by the pad, while the plasmaflows through readily. It is believed that the agglutinating agent bindsred blood cells, forming agglutinates thereof, while the nucleatingparticles provide a nucleus for formation of clusters of theagglutinates. However, it should be understood that the practice of thisinvention is not dependent on the correctness of this theory.

For the purpose of the present invention, "substantially completeremoval" of red blood cells means that the red blood cells are removedfrom the sample such that there is virtually no interference with anysubsequent assay performed on the sample.

In one embodiment of the present invention, high molecular weightpolyethylene glycol (MW>10,000) is added to the combination ofagglutinating agent and nucleating particles. The polyethylene glycolenhances the formation of clusters.

The blood separation means of the present invention has the followingfeatures and advantages:

(1) Since whole blood can be directly used as a sample for assay, theprocessing operation is very simple and convenient. There is norequirement to separate the red blood cells from the sample prior toconducting the assay. Furthermore, the preparation time up to thechemical analysis per se is very short. Despite this, the red blood cellseparating means of this invention can permit dry-chemical analysis ofblood with accuracy equivalent to wet-method chemical analysis ofcomponents dissolved in blood using serum or plasmas a sample.

(2) The sample fluid resulting from the filtration of formed componentsfrom the whole blood transferred to the porous material is suppliedsmoothly to the analysis area of an assay device. Because the wholeblood travels through the absorbent pad and secondary filter ratherrapidly and most of the liquid traverses the red blood cell removingmeans, an accurate determination can be made of the analyte of interest.

In a preferred embodiment, a flow delay means is provided in the deviceto delay the flow of the fluid sample after the red blood cells havebeen removed. This permits incubation of the fluid sample for prolongedcontact with an enzyme or chemical reagent where necessary. The flowdelay device inhibits sample flow for a predetermined discrete period oftime, after which the sample completely flows through the flow delaymeans for further processing.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a device according to the invention.

FIG. 2 shows a device according to the invention with an optionalsecondary filter.

FIG. 3 shows the device according to the invention incorporated in adiagnostic device.

FIG. 4 is a schematic showing the operation of the device of FIG. 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Blood is composed of two parts: (a) plasma, the fluid portion; and (b)formed elements, the blood cells and platelets suspended in the fluid.The plasma accounts for about 55% of the total volume of blood, and isabout 92% water, 7% protein, and less than 1% other substances. Theprincipal plasma proteins are the globulins, albumin, and fibrinogen.Plasma from which fibrinogen has been removed is called serum. Thesuspended particle include erythrocytes (red blood cells), leukocytes(white blood cells), and thrombocytes (platelets). Most of the bloodcells (corpuscles) are red blood cells. White blood cells are largerthan red blood cells, but fewer in numbers.

For purposes of the instant invention, the term "whole blood" indicatesplasma which still contains a substantial number of red blood cells,even if some red blood cells had previously been removed. The removal ofred blood cells from a whole blood sample is a form of bloodfractionation. Blood fractionation is important in clinical chemistrybecause chromogenic analysis is generally used to determine theconcentration of particular components dissolved in blood. The presenceof a particular component is signaled by color production or colorchange. The presence of red blood cells in whole blood interferes withthe reading of the chromogenic analysis due to the turbidity and colorof the whole blood. Therefore, it is important to remove red blood cellsfrom whole blood prior to using the remaining serum or plasma in theanalysis of dissolved blood components.

Thus, it is desirable to provide means for removing substantially all ofthe red blood cells from a sample of whole blood. For purposes of thisinvention, a sample is considered to be substantially free of red bloodcells when any red blood cells that may remain in the sample do notinterfere with the accuracy of the analysis for the analyte sought to bedetected in the sample. That is, any red blood cells that may remain inthe sample are not detectable by or do not interfere with the analyticmethod employed for detection of the analyte of interest.

Cluster Forming Means

The cluster forming means comprises any suitable material through whichplasma or serum can pass, which is inert to whole blood and which doesnot interfere with subsequent assays of the blood sample, in combinationwith at least one agglutination agent and nucleating particles. Aparticularly useful cluster forming means is an absorbent pad whichcontains a combination of agglutinating chemicals and nucleatingparticles. Pads made from materials such as glass or synthetic fiberscan be used. The pads can be woven or nonwoven. The fibers arepreferably hydrophilic, either by the nature of the fibers themselves orby post treatment of the fibers. The fiber size is not material.

The clusters formed range in size from about 100 to 1500 microns. Mostclusters fall into the range of from about 250 microns to 1000 microns.

The mesh or pore size of the pad should average about 200 microns, witha preferred range being from about 20 to about 500 microns.

One particularly suitable material for the absorbent pad is Orlon®fibers in a pad approximately 1/8 inch thick, with an average pore sizeof about 200 microns. Agglutinating chemicals and nucleating particlesare incorporated into the absorbent pad by impregnation thereof, and thepad is subsequently dried. Optionally, an anticoagulant is used in theabsorbent pad, e.g., EDTA or sodium heparinate.

In another embodiment, high molecular weight polyethylene glycol is alsoincorporated in the pad. Table 1 shows ranges for combinations ofagglutinating agents and nucleating particles that can be used in thepresent invention. In Table 1, the rate of agglutination was rated from1 to 4, with 4 being the fastest and 1 being the slowest. Theconcentration of each component is expressed in terms of theimpregnating solution. Lectin is present in each experiment.

                  TABLE 1                                                         ______________________________________                                        1a) Effect of lectin concentration (Phaseolus vulgaris) on                    agglutination rate                                                            Lectin conc.                                                                             0      100    200   300   400                                      (mg/mL)                                                                       Agglutination rate                                                                       1      3      4     4     4                                        1b) Effect of nucleating particle(polyacrolein/iron oxide)                    concentration on agglutination rate                                           Particle conc.                                                                           0      22     44    66    88                                       (mg/mL)                                                                       Agglutination rate                                                                       2      4      4     4     4                                        1c) Effect of molecular weight of PEG on agglutination rate                   MW of 5%,  6000   8000   10000 20000 35000 40000                              w/v PEG                                                                       Agglutination rate                                                                       1      1      2     4     4     4                                  ______________________________________                                    

The concentrations of agglutinating agent and nucleating agent arechosen so as to form clusters from substantially all of the red bloodcells in a sample. Typically, the agglutinating agent will be present ina concentration ranging from about 10 to about 4500 micrograms/mL, andthe nucleating particles will be present in a concentration of fromabout 0.1 to about 20 mg/mL. Where optional polyethylene glycol is usedwith the agglutinating agent, the polyethylene glycol is used inconcentrations of approximately 0.1 to 30% w/v solution.

A buffered solution may be used to impregnate the absorbent pad with theagglutinating agent and nucleating particles and, where used,polyethylene glycol. This buffer may include phosphate buffered saline,tris MOPS, borate, carbonate, or the like. Usually, the solution isbuffered to a pH in the range of about 4 to 9. The concentrations ofagglutinating agent, nucleating particles, and polyethylene glycol inthe buffered solution are about 10 to 400 micrograms/mL, 0.1 to 20mg/mL, and 0.1 to 30% w/volume, respectively.

In the case of cholesterol assay, as illustrative of other assays, theimpregnating solution has from about 2 to 100 units/ml of the twoenzymes, cholesterol esterase and cholesterol oxidase. The detergents,if used, have a total weight of from about 0.1 to 5 weight percent ofthe medium. The binding agents or adhesives are generally in the rangeof about 0.2 to 10 or more usually from about 1 to about 5 weightpercent of the total detergent mixture. A preservative or hydrogenbonding agent may be present in from about 1 to 20 weight percent moreusually from about 2 to 10 weight percent. The remaining additives aregenerally present in a total amount of less than about 10 weightpercent. The remaining composition may be water, nonreactiveingredients, excipients, extenders, and the like.

Agglutinating Agents

Although many agglutinants for red blood cells are known, theagglutinants used must be such that they do not interfere with anysubsequent analysis. Preferably, the agglutinating agents have fastaction and a short reactivation time from the dry to the wet state, arenon-specific to blood types and are stable and inexpensive.Agglutinating agents can readily be selected by screening tests withwhole blood samples, which is well within the skill of the art.

The preferred agglutinants are lectins, including concanavalin A, wheatgerm agglutinin, and the agglutinins of Glycine max. and Phaseolousvulgaris, either separately or in combination. Lectins are protein,widely distributed in nature, which are able to agglutinate erythrocytesand many other types of cells. More specifically, the term "lectin"denotes "a sugar-bonding protein or glycoprotein of non-immune originwhich agglutinates cells and/or precipitates glycoconjugates," Goldsteinet al., Nature 285:66 (1980). Lectins occur primarily in plant seeds,but the also occur in roots, leaves and bark. In addition, lectins arepresent in invertebrates such as clams, snails, and horseshoe crab, andin several vertebrate species.

Members of the lectin family include concanavalin A, arbin, ricin, aswell as soybean agglutinin and wheat germ agglutinin. Further backgroundon lectins can be found in H. P. Schnebli and J. Bachi, "Reactions ofLectins with Human Erythrocytes," Exot. Cell. Research 91 (1975); and"Lectins and Lectin Conjugates'" a general lectin catalogue produced byEY Laboratories, Inc., San Mateo, Calif., 1992.

Alternatively, antibodies can be used as agglutinating agents. Theantibodies used have a binding affinity for a determinant present on thesurface of red blood cells. Antibodies reactive with any antigen presenton the surface of a red blood cell can be used, including but notlimited to major histocompatibility antigens, cell surface proteins,cell surface carbohydrates, and cell surface glycoproteins. Antibodiesfor agglutinating erythrocytes are well known, and may be based uponantibodies which recognize various antigenic surface constituents of theerythrocytes, including proteins, glycoproteins, glycolipids, andlipoproteins. Antibodies which recognize these constituents may beprepared by conventional techniques using the membrane, or the purifiedconstituents themselves, as immunogens. These antibodies may bemonoclonal or polyclonal in nature. Either the intact antibody, orspecific binding fragments thereof, may be used as agents to agglutinatethe erythrocytes.

It has also been discovered that polyethylene glycol can be added to thecombination of the agglutinating agent and nucleating particles toenhance formation of clusters from red blood cells. As shown in Table 1above, the agglutination rate is enhanced by the addition ofpolyethylene glycol having a molecular weight about 10,000. It was foundthat polyethylene glycol having a molecular weight of about 35,000 wasparticularly suitable, but that polyethylene glycol having molecularweights about 35,000 can also be used. The polyethylene glycol can beincorporated in the pad in the same manner as the agglutinating agentand nucleating particles, preferably by impregnation from a solutionthereof.

Further discussion of erythrocyte agglutinating and binding material isgiven in Hillyard et al., U.S. Pat. No. 5,086,002, which patent ishereby incorporated by reference in its entirety.

It is desirable that the agglutinating agent be capable of agglutinatingall red blood cells of the species, e.g., human, to which the subjectbelongs. If a single monoclonal antibody is not available which is notserospecific, one may use a polyclonal antiserum against human red bloodcells, or a defined mixture of monoclonal antibodies that collectivelyreact with all of the major blood types. The antibody can be coated ontothe nucleating particle, or can be slurried or dissolved in a suitableliquid for impregnation into the absorbent pad. There is generally aminimum amount of antibody that must be used in the blood separationdevice in order to remove substantially all of the red blood cells fromthe whole blood. However, it is not possible to give a specific amountof an antiserum that must be used, since different antisera differ intheir ability to bind red blood cells. Accordingly, the optimum amountof antibody is determined empirically, which can readily be effected byone skilled in the art. Serial two-fold dilutions of neatantibody.containing solution or antiserum are applied to filters alongwith the desired nucleating agents in an amount sufficient to saturatethe filter. Efficiency of filtration, lysis of red blood cells, andamount of plasma that passes through the filter when a standard amountof whole blood is applied are measured.

Other types of agglutinating agents for red blood cells includepolymeric amino acids such as polylysine, polyarginine, and the like.

Nucleating Particles

The nucleating particles for use in the present invention are particleswhich are capable of acting as nucleating agents and which do not reactwith any of the components of the blood to interfere with subsequentassays. These particles have an average diameter of from about 0.1micron to about 100 microns, and preferably less than about 10 micronsin diameter. When the particles are much greater in diameter than 100microns, it is difficult to impregnate the particles into the padmatrix. Ideally, the nucleating particles are the same size or smallerthan the red blood cells themselves.

Particles useful in the present invention are those which absorb oradsorb lectins or other agglutinating agents on the surfaces of theparticles. For example, the porous iron oxide particles used herein aremagnetizable polyacrolein beads which contain iron oxide. These beadshave reactive aldehyde groups on the surface thereof which link withlectins upon contact. Alternatively, the lectin or other agglutinatingagent can be chemically immobilized onto the surface of the nucleatingparticles. Non-limiting examples include lectin which has beenimmobilized onto silica, lectin which has been immobilized into glassbeads, or lectin immobilized onto agarose. These nucleating particleswith agglutinants on their surfaces thus allow red blood cells toagglutinate around them to form large clumps, or clusters.

Many other inert particles that permit absorption or immobilization oflectins or other agglutinating agents on their surfaces can also be usedas nucleating particles in the present invention. Among these particlesare latex beads, glass beads, cellulose, titanium dioxide, polyacrolein,alumina, iron oxide, chromium oxide and the like.

For the purposes of the present invention, the preferred nucleatingparticles are polyacrolein or polyacrolein/iron oxide beads. However, asshown in Table 2, many types of particles can be used, and the inventionis not limited to any specific particles. Additionally, combinations ofnucleating particles can be used, in any desired proportions.

                  TABLE 2                                                         ______________________________________                                        Relative agglutination rate enhancement of                                    different nucleating particles                                                Particle            Relative agglutination rate                               ______________________________________                                        free lectin         1                                                         free lectin + silica                                                                              1                                                         lectin attached to polyacrolein bead                                                              2                                                         lectin attached to 0.3 micron latex                                                               20                                                        ______________________________________                                    

The Secondary Filter

Optionally, a secondary filter is used to trap any extra red blood cellsthat may escape from the absorbent pad. This secondary filter my be madeof any filtration material which is compatible with the assay and whichis capable of separating red blood cells from plasma. For example, itcan be made of glass fiber paper, filter paper, or synthetic filtermaterials such as porex filter. The secondary filter should ideally havea very small pore size to permit plasma to pass while retaining anyresidual red blood cells. Ideally, a porous matrix with pore sizesbetween 1 and 5 microns can be used. A glass fiber filter with a meanpore size of 1.2 microns and a thickness of 737 microns is preferred,although a much thinner porous filter can also be used. Conveniently,when a secondary filter is provided, it is configured as the secondlayer of a two layer device.

Flow Delay Coating

The present invention also optionally provides a coating on a substrateto inhibit flow of a liquid through the substrate for a predeterminedperiod of time. After a predetermined period of time, the liquid iscompletely released through the substrate. There is substantially noeffect on the viscosity and flow characteristics of the fluid. The rateof flow delay, i.e., the length of time the liquid is retained on thesubstrate, is controlled by the amount of coating applied to thesubstrate. The control means is self-regulating.

For example, a coating is applied to a pad made of paper, glass fiber,fabric, or similar materials to provide flow-control means for use indiagnostic devices. These pads retain fluid for a discrete period oftime to enable substances in the fluid to react with components in thepad for a discrete period of time, after which the fluid flowscompletely through the pad. Different types of reactions that requiredifferent incubation times can all use the same type of pad withdiffering levels of coating applied to correspond to the desired delaytime. The coating materials do not chemically affect the reagentmixture, nor do they affect the viscosity of the sample or reagentfluid, and thus do not affect the flow of the reagent fluid or the runtime of a reagent strip portion of an assay device.

For the purpose of the present invention, "flow delay" means that theflow of a fluid through a material is delayed for a certain length oftime, after which the fluid flows substantially completely through thepad. For example, for the critical period of time_(c), from introductionof the sample, t_(a), to t_(c), the flow rate is such that in this timeperiod no more than 10%, and preferably no more than 5%, of the sampleflows through the material. After t_(c), substantially all of theremainder of the sample passes through the flow delay material.Moreover, the rate of flow after t_(a) should be substantially greaterthan the rate of flow before t_(a), as the sizing material does notincrease the viscosity of the sample.

As different reactions require different incubation times, t_(c)preferably ranges from 2 seconds to twenty minutes. t_(c) morepreferably ranges from 2.5 seconds to about 5 minutes. Particularly forquantative assays, the amount of sample retained in the flow delaymaterial must be a sufficient amount of sample to be incubated in theflow delay material to effect a valid assay. Likewise, the amount ofsample which passes through the flow delay material must be a sufficientamount of sample to effect a valid assay.

The flow delay means of the present invention, in other words, acts likea timed valve, in that the fluid is retained in the flow delay means fora predetermined time, t_(a). After this time t_(c), the fluid flowssubstantially completely through the flow delay means, such as into anadjoining layer of an assay device.

Once the flow delay means has "opened" to the fluid, at t_(c), it isimportant that substantially all of the fluid flow through the flowdelay means rather than remain in the flow delay means. The flow delaymeans retains the fluid in the flow delay area only for a predeterminedtime, and then permits the fluid to flow through the flow delay meanssubstantially without changing the fluid. The flow delay means does notaffect the viscosity of the fluid, nor does the flow delay means addextraneous material to the fluid sample from the flow delay means. Thsfluid is free to flow directly through the flow delay means just as if amechanical valve had been opened, and substantially all of the fluidflows through this "valve" without being retained in the flow delaymeans.

One way of expressing the fluid flow through the flow delay means is interms of the time required for the sample to pass completely through theflow delay means once the predetermined time, t_(c), has passed. Thistime period from initial contact of the fluid sample with the flow delaymeans, t_(a), to the predetermined time, as noted above, can beexpressed as t_(a). The time required for the sample to flow through theflow delay means after t_(c) can be expressed as t_(f). Ideally, t_(f)should not be much greater than t_(c), and may often be smaller thant_(a), depending upon the length of time the sample is retained by theflow delay device, the nature of the sample, and subsequent operationsto be performed on the sample.

For example, if t_(c) is one minute, t_(f) can be from less than oneminute to greater than five minutes, depending upon the initialviscosity of the sample, the rapidity with which the test must beconducted, and the like. On the other hand, if t_(a) is 20 minutes, itmay be useful for t_(f) to be less than 20 minutes. Depending upon thetype of sample applied to the flow delay means and the rate at which onedesires the sample to flow through the device, one can adjust t_(c) andt_(f) so that they are multiples of each other. One skilled in the artcan readily determine the relationship between t_(c) and t_(f) which isoptimum for the particular fluid sample to be treated, and can adjustthe flow delay means accordingly.

In another example, with a relatively viscous fluid sample which onlyrequires an incubation of one minute, t_(c) can be one minute and t_(f)can be up to about ten minutes. On the other hand, for a relativelyfree-flowing sample which requires a long incubation period, such as 20minutes, t_(f) can be a fraction of t_(a), i.e., 5 minutes, or 1/4t_(a). The relationship between t_(c) and t_(f) is not critical to theinvention; the criticality is that the flow delay means act as a meansto retain the fluid sample for a predetermined time, and then releasethe sample through the flow delay means.

A great many materials can be used to control the flow of liquidsthrough substrates, depending upon the type of fluids used. Examples ofthese materials include alkyl ketene dimers, alkenyl succinicanhydrides, and fluorocarbon resins, either alone or with othermaterials such as saccharides, low molecular weight polymers, waxes andthe like. Since not all materials offer equivalent degrees of fluidretention, the material used to coat or impregnate the substrate can bechosen to provide the desired flow delay time for the fluid to becontrolled. These coating materials for flow delay are designated"sizing materials" for the purpose of the present invention. A number ofsizing materials can be applied to fibrous substrates, such as pads,which retain fluids in the substrate for a predetermined discrete periodof time. This time period can range from less than one minute to morethan 20 minutes, depending upon the fluid, the nature of the sizingmaterial, and the amount of sizing material applied to the substrate.

The flow control means of the present invention has the followingfeatures and advantages:

The degree of flow control means is readily metered by the amount ofsizing material applied to the substrate.

The sized substrates are self-regulating, and require no designed-inmoving parts and no input from the user for accurate operation.

Different types of reactions that require different incubation times canall use the same type of pad with differing levels of sizing materialapplied corresponding to the desired flow delay.

The delay times obtained are highly reproducible.

The sizing materials used for flow delay are inexpensive and are easilyand accurately dispensed onto the substrate material.

The sizing material do not chemically affect reagents used in clinicalassays.

The sizing materials used do not affect the viscosity of the reagentfluid or the sample and thus do not affect the flow of the reagent fluidor the run time of the reagent strip portion of the device.

Some of the sizing material can be covalently attached to the substrate,particularly if the substrate is paper or a surface-modified glass fibermaterial.

By putting a flow delay coating on the secondary filter pad, the samplecan be retained in the primary filter pad for an extra 5-10 seconds, sothat the whole blood sample can be incubated with lectins and otherchemicals. While the whole blood is maintained in the primary filter padfor the extra incubation period, most of the large particles formed aretrapped in the ABS pad, and few or no red blood cells enter the bottomof the secondary filter pad. This time delay mechanism worked very well,and a delay of as little as 5-10 seconds actually helped to reduce thetotal flow time through the device by more than 25%. In addition, no redblood cell leakage was observed.

Reagent Means

The red blood cell separating means can be used with any assay devicewhich includes a reactant pad through which fluid flows to produce adetectable signal. After addition of the sample to the reactant padthrough the red blood cell separation means, and an incubation of UD toabout 30 minutes, the analyte reacts with the reagents in the reactantpad to produce a detectable signal.

The red blood cell separation device can, of course, be used with assaydevices for a great variety of substances in the blood in addition tocholesterol, including glucose, blood urea nitrogen, uric acid, albumin,creatinine, bilirubin, phosphate, total protein, amylase, calcium, etc.

The red blood cell removing means of this invention can be used with anytype of assay that can be conducted using a reactant or reagent zone forreaction with the analyte of interest. A variety of sophisticatedreagents, protocols or regimens can be devised based upon a limitedamount of material migrating to produce a boundary in proportion to theamount of material present. Examples of protocols include particleshaving first and second ligands, where the first ligand competes withanalyte for receptor bound to a surface. After carrying out competitionfor a limited amount of receptor between analyte and sample pad and theparticle transported with the effluent through the measurement region.By having receptor for the second ligand in the measurement region, theparticle boundary will be defined by the number of particles added tothe pad. By having colored particles, charcoal particles, magneticparticles, dyes, dye-polymer conjugates, protein with high visibleextinction coefficients, etc., the boundary will be readily defined.

Any technique which allows for binding of a detectable entity inproportion to an analyte of interest may be employed. These may includecleavage of a bond to release the entity, where the bond to the entityis not clearable when the entity is bound to a receptor, binding to asupport which inhibits migration of the entity in proportion to theamount of analyte in a sample, or the like. The entity may be a particleas described above, an enzyme which catalyzes the production of adetectable product, or the like.

Of particular interest is where a product is produced on the sample padwhich provides for a detectable boundary. For example, where the analyteis an enzyme substrate, the sample pad may be impregnated with theappropriate enzyme or enzymes to produce a product. Normally, the enzymeproduct will react, either directly or indirectly, with a compound whichis fixed in the assay measurement region. This may be exemplified bycholesterol, glucose, or the like, which reacts with an oxidase toprovide an oxidizing species. The oxidizing species may then react withthe bound compound to produce a detectable boundary. Illustrative ofthis situation would be the hydrolysis of serum cholesterol ester bycholesterol esterase and subsequent oxidation of cholesterol bycholesterol oxidase to produce a stoichiometrically identical amount ofhydrogen peroxide. This hydrogen peroxide is formed at a stationaryreaction pad and combines with horseradish peroxidase which is in themobile phase. The horseradish peroxidase/hydrogen peroxide reacts with abound substrate to produce a detectable boundary.

Depending upon the assay, other reagents may also be present. Forexample, detergents can be used where a lipophilic analyte in blood isinvolved, where the lipophilic analyte binds to proteins bound to blood.This may be illustrated by cholesterol which binds to proteins, as forexample, in very low, low and high density lipoproteins. Thus,detergents such as nonionic, anionic or cationic detergents may be used.Of particular interest are polyoxyalkylene, ethoxylated alkylphenol,octylphenoxypolyethoxyethanol, octylphenol-ethylene oxide condensatesand polyoxyethylene lauryl ether, or anionic detergents, such as bileacids, e.g., sodium cholate and sodium taurocholate. In addition,various sticking agents or adhesives which are substantiallynoninterfering, which may include gelatin, casein, serum albumin, orgamma globulins may be incorporated in the reagent pad. Also, thereagent pad may include preservatives, such as sucrose, polyvinylalcohol, polyvinyl pyrrolidone, dextran, as well as catalase inhibitorssuch as sodium azide or hydroxylamine salts.

The Apparatus and Method

Red blood cells are rapidly removed from a sample of whole blood usingthe blood separation device 10 of the present invention as shown inFIG. 1. Whole blood is introduced to the absorbent pad 1, whichabsorbent pad is impregnated with an agglutinating agent and anucleating agent. Most of the separation of red blood cells occurs inthe absorbent pad, where the red blood cells are agglutinated into largeclusters which are retained in the absorbent pad. The blood then rapidlypasses into the secondary filter 2 for final separation of any left overred blood cells that may remain in the sample. Since white blood cells,which only occupy about 1% of the total volume of blood, are much largerthan red blood cells, the white blood cells are trapped by the filter.

FIG. 1 illustrates one embodiment of the invention. When whole blood isintroduced into the absorbent pad 1, the red blood cells contact theagglutinating agent and nucleating particles and agglutinate veryquickly, e.g., in less than 5 seconds, into large clumps of cells. Theclumps have an average diameter of about 500-1000 microns. The red bloodcell aggregates remain in the absorbent pad because the pore size of thepad is small enough to retain the aggregates. The plasma, which is nowpredominantly red blood cell free plasma, flows into the secondaryfilter 2. The secondary filter 2 traps any extra red blood cells thatescape from the absorbent pad.

FIG. 2 shows an optional third layer 3 which has an agglutinating agentdeposited thereon. This third layer is present to ensure a plasmasubstantially free of red blood cells for particularly sensitive assays.

FIG. 3 shows an assay device 30 incorporating the blood separation meansof the present invention. Whole blood 31 is introduced to the devicethrough the absorbent pad 32. This absorbent pad is impregnated withbeads of acrolein/iron oxide which have been coated with a lectin. Mostof the red blood cells in the sample agglutinate almost immediately uponcontact with the absorbent pad, and are retained in this part of thedevice. Blood which is substantially free of red blood cells flows tothe secondary separation pad 33. This pad is made of 1.2 micron glassfibers, and traps any red blood cells that were not agglutinated andretained in the absorbent pad. The sample then flows to the enzyme pad34 that contains the cholesterol enzymes (cholesterol esterase andcholesterol oxidase). After cholesterol in the sample is converted tohydrogen peroxide, the sample passes through pad 35, the time delay pad,and finally contacts the measurement dye zone 36. The measurement dyezone 36 is coated or impregnated with an indicator material that reactswith the enzyme-treated sample to give an indication of the presence oramount of analyte in the sample.

FIG. 4 illustrates use of the device according to the present inventionfor a cholesterol determination. Of course, the device can be used witha great variety of clinical assays, and this illustration is notintended to limit the invention to one type of assay.

Referring to FIG. 4, a sample of whole blood, generally approximately100 micrometers, is introduced into an absorbent pad 1. The red bloodcells present in the whole blood agglutinate into large (500-1000micron) clusters 40 around nucleating particles and are trapped in theabsorbent pad. Any small clumps 41 that leak through the absorbent pad 1with the plasma are trapped by the secondary glass fiber pad 2. Plasmathen enters the enzyme pad 4. The enzyme pad is impregnated withcholesterol esterase and cholesterol oxidase. The cholesterol in theplasma reacts with these enzymes to produce hydrogen peroxide.

The hydrogen peroxide in the plasma then flows into the measurement zone5 that contains a dye immobilized thereon. This dye reacts withhorseradish peroxidase to form a color bar 6, the length of which isrelated to the amount of cholesterol in the sample.

The following examples illustrate the capacity of the device of thepresent invention to remove red blood cells from whole blood. Theseexamples are included for purposes of illustration and comparison only,and are not intended to limit the scope of the invention.

EXAMPLE 1

The device shown in FIG. 1 (10) has an absorbent pad 1 made of Orlon®,fibers from Lydall Westrex with dimensions of 9.0 mm×6.1 mm×2.1 mm. Asolution of 125 U/mL lectin in 5 mM phosphate buffered saline, pH 7.2,was made up. Seventy microliters of this solution was mixed with 1.4 mgof polyacrolein/iron oxide particles of size about 1-20 microns, with amean particle size of 10 microns. The mixture was added to the pad,which absorbed the liquid. The pad was air dried for twelve hours at 70°F. and 5% relative humidity.

A secondary filter (2) was made from a glass fiber Whatman GD1 filtermaterial cut to dimensions of 7.2 mm×5.5 mm×1.4 mm. This secondaryfilter was placed below the absorbent pad.

The device as prepared above was tested by depositing 140 microliters ofwhole blood having a hematocrit of 40-50% red blood cells onto theabsorbent pad. The effectiveness of separation of red blood cells fromwhole blood was tried with a variety of agglutinating agents andnucleating agents. The speed of separation, the amount of plasmarecovered, and the amount of red blood cell leakage are shown inTable 1. A rating of 0 RBC leakage represents no red blood cell leakage,and the plasma sample is visibly clear. A rating of 4 represents thatthe plasma recovered contained more than 20% red blood cells.

Conventional glass fiber pads do a poor job of separating 140microliters of whole blood, as shown in row 1 of Table 2a. Adding freelectin improved the plasma yield while also shortening the separationtime. Lectin with nucleating particles (row 3) and lectin immobilized onsilica (row 4) were found to be the most effective in giving clean,efficient, and fast plasma separation.

                  TABLE 2a                                                        ______________________________________                                                     YIELD                                                                         OF                                                                            PLASMA SEPARATION RBC                                                         (uL)   TIME (sec.)                                                                              LEAKAGE                                        ______________________________________                                        1.  ABS.PAD + GD1  15       192      4                                        2.  FREE LECTIN (GD1)                                                                            25       25         0.5                                    3.  LECTIN + BEAM  32        5       0                                            (GD1)                                                                     4.  LECTIN ON SILICA                                                                             34       42       1                                            (GDI)                                                                     ______________________________________                                    

EXAMPLE 2

A separation device 20 as shown in FIG. 2 was prepared having anabsorbent pad 1 made from Orlon®, fibers from Lydall Westex withdimensions of 9.0 mm×6.1 mm×2.1 mm. A solution of 125 u/mL lectin in 5mM phosphate buffered saline, pH 7.2, was made up. Seventy microlitersof this solution was mixed with 1.4 mg of acrolein/iron oxide particlesand the mixture was added to the pad, which absorbed the liquid. The padwas air dried for twelve hours at 70° F. and 5% relative humidity.

A secondary filter (2) was made from Whatman GD1 filter material cut todimensions of 7.7 mm×5.5 mm×0.5 mm. This secondary filter was placedbelow the absorbent pad.

A third filter (4) was placed below the secondary filter. This thirdfilter also had dimensions of 7.7 mm×5.5 mm×0.5 mm.

The absorbent pad in each trial was made of S&S Grade 5.S nonwoven rayonpads, S&S Grade 40 glass fiber pads, or S&S grade 404 filter paper. Eachabsorbent pad was impregnated with 3 micrograms of free lectin in 0.5 mMPBS, pH 7.2 and air dried prior to assembling the device.

The devices were tested by depositing 140 microliters of whole bloodhaving a hematocrit of 40-50% red blood cells onto the absorbent pad.Leakage of red blood cells are shown in Table 3, demonstrating thatabsorbent pads made of fibers other than glass were as effective ascorresponding absorbent pads made of glass fibers.

                  TABLE 3                                                         ______________________________________                                                       Yield of                                                                             Separation                                                                              RBC                                                          Plasma Time (sec.)                                                                             Leakage                                       ______________________________________                                        ABS.PAD + S&S40 + S&S5 - S                                                                     40       20        0                                         ABS.PAD + S&S40 + S&S404                                                                       29       19        0                                         ABS.PAD + S&S5 - S + S&S404                                                                    26       20        0                                         ______________________________________                                    

EXAMPLE 3

In this example, lectins purified from Phaseolus vulgaris were used asin Example 1 to impregnate the absorbent pad. Lectins purified fromPhaseolus vulgaris were found to be the most effective and the mostnon-specific for different blood types. However, mixtures of two or moretypes of lectins have also been found useful in separating red bloodcells. In all cases, lectins in combination with nucleating particlesdecreased the separation times by 3 to 5-fold when compared with theresults with free lectins alone. The absorbent pads and the nucleatingparticles used were the same as in Example 2.

                  TABLE 4                                                         ______________________________________                                                      Yield of Separation                                                                              RBC                                                        Plasma (μL)                                                                         Time (sec.)                                                                             Leakage                                      ______________________________________                                        LECTIN     TYPE O   25         25        0.5                                  LECTIN + BEAD                                                                            TYPE O   32          5      0                                      LECTIN     TYPE A   30         34      0                                      LECTIN + BEAD                                                                            TYPE A   39          7      0                                      LECTIN     TYPE AB  26         25      0                                      LECTIN + BEAD                                                                            TYPE AB  34          6      0                                      ______________________________________                                    

EXAMPLE 4

Using the assay device 30 shown in FIG. 3, two drops of whole blood froma finger stick 31 (approximately 100 microliters) were placed onto acluster forming means comprising an absorbent pad 32 which hadpreviously been impregnated with iron oxide particles and lectin as inExample 2. The red blood cells in the whole blood were agglutinated intoclusters in the absorbent pad 32 and rapidly passed through secondaryfilter 33 and tertiary filter 34 to remove substantially all of the redblood cells from the sample. This agglutination/filtration wasaccomplished in less than 10 seconds. The blood then contacted a pad 35which had been impregnated with an enzyme which uses the analyte ofinterest as a substrate to form hydrogen peroxide, and the sample thencontacted the measurement zone 36. As the hydrogen peroxide formed fromthe analyte in the sample entered the measurement zone 36, a color bardeveloped. The length of this color bar was proportional to theconcentration of analyte in the sample.

Analytes which can be determined in a device according to this exampleare particularly those which form hydrogen peroxide upon contact withthe appropriate enzyme. Among these analytes are cholesterol, glucose,uric acid, and the like.

EXAMPLE 5

Preparation of the Flow Delay Coating

One gram of palmitoyl ketene dimer was dissolved in 25 mL ethyl acetate.This stock solution was diluted to appropriate levels, i.e., to a levelthat permits mechanical dispensing of the solution into pads in aprecise manner. Six micrometer aliquots of a chosen dilution wereapplied to pre-cut secondary filter pads. The pads were dried in a 100°C. oven for 30 minutes and cooled in a desiccated atmosphere. The padswere installed in the device of Example 2 as a secondary filter (2).

EXAMPLE 6

    ______________________________________                                        Effect of flow delay on red blood cell leakage                                Amt. of flow                                                                  delay coating                                                                             Approx. flow                                                      per pad     delay time   Red blood cell leakage                               ______________________________________                                        0 μg     0 sec.       slight                                               4           <5           very slight                                          5           5-10         none                                                 6           >10          none                                                 ______________________________________                                    

EXAMPLE 7

    ______________________________________                                        Effect of Flow Delay Pad on Flow Rate                                                         Ave. Time Ave. Total                                          Geometry        through pads                                                                            flow time                                           ______________________________________                                        no flow delay   34 sec.   18.6 min.                                           with flow delay 42 sec.   14.2 min.                                           ______________________________________                                    

When the flow delay pads of 5 μg/pad were tested against pads withoutflow delay the average flow time through the front end pads in thedevices was delayed by less than 10 seconds, but the total flow timethrough the device actually decreased by 4 minutes. Thus, using the flowdelay means in the present invention provided a more efficientseparation of red blood cells.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without departing from the generic concept,and therefore, such adaptions and modifications should and are intendedto be comprehended within the meaning and the range of equivalents ofthe disclosed embodiments. It is to the understood that the phraseologyand terminology employed herein is for the purpose of description andnot of limitation.

All patents, patent applications, and other literature cited herein arehereby incorporated by reference in their entirety.

What is claimed is:
 1. Apparatus for separation of red blood cells from whole blood comprisinga cluster forming means comprising a material through which plasma or serum can pass which is impregnated or coated with a mixture of a free first red blood cell agglutinating agent and nucleating particles to which said first red blood cell agglutinating agent is conjugated.
 2. Apparatus according to claim 1 wherein said material through which plasma or serum can pass is an absorbent pad.
 3. Apparatus according to claim 1 further comprising a filter located under said cluster forming means.
 4. Apparatus according to claim 3 wherein said filter contains a second agglutinating agent, wherein said second agglutinating agent may be the same or different from said first agglutinating agent.
 5. Apparatus according to claim 1 wherein said first agglutinating agent comprises at least one lectin.
 6. Apparatus according to claim 1 wherein said nucleating particles are selected from the group consisting of particles or beads of polyacrolein, polyacrolein/iron oxide, latex, silica, glass, agarose, and mixtures thereof.
 7. Apparatus according to claim 6 wherein said nucleating particles are particles of polyacrolein iron oxide.
 8. Apparatus according to claim 6 wherein said nucleating particles are particles of latex.
 9. Apparatus according to claim 3 wherein said filter includes flow-delay means, wherein said flow delay means comprises a coating of a sizing material on a substrate to inhibit flow of a liquid through the substrate for a predetermined period of time.
 10. Apparatus according to claim 9 wherein said flow delay means comprises a coating of a flow-delay substance selected from the group consisting of alkyl ketene dimers, alkenyl succinic anhydrides, fluorocarbon resins, and mixtures thereof.
 11. Apparatus according to claim 10 wherein said flow-delay substance further comprises at least one substance selected from the group consisting of saccharides and polymers. 